Compositions and Methods for Inhibiting Viral Replication

ABSTRACT

The present invention relates to a double-stranded ribonucleic acid (dsRNA) having a nucleotide sequence which is less that 30 nucleotides in length and which is substantially identical to at least a part of a 3′-untranslated region (3′-UTR) of a (+) strand RNA virus, such as HCV, as well as pharmaceutical compositions comprising the dsRNA, together with a pharmaceutically acceptable carrier. The pharmaceutical compositions are useful for treating infections and diseases caused by the replication or activity of the (+) strand RNA virus, as well as methods for inhibiting viral replication.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/959,936, filed Dec. 19, 2007, which is a divisional of U.S. patentapplication Ser. No. 10/384,512, filed Mar. 7, 2003, (which issued asU.S. Pat. No. 7,348,314 on Mar. 25, 2008) which is acontinuation-in-part of International Application No. PCT/EP02/11432,which designated the United States and was filed on Oct. 11, 2002, whichclaims the benefit of German Patent Application No. 101 50 187.0, filedon Oct. 12, 2001, German Patent Application No. 101 55 280.7, filed onOct. 26, 2001, German Patent Application No. 101 58 411.3, filed on Nov.29, 2001, German Patent Application No. 101 60 151.4, filed on Dec. 7,2001, German Patent Application No. 101 63 098.0, filed on Dec. 20,2001, International Application No. PCT/EP02/00151, filed on Jan. 9,2002, and International Application No. PCT/EP02/00152, filed on Jan. 9,2002. The entire teachings and contents of the above applications areincorporated herein by reference, including any appendices orattachments thereof, for all purposes.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use for inhibiting the replication of (+) strand RNA viruses, suchas Hepatitis C virus, as well as treating viral-associated diseases.

BACKGROUND OF THE INVENTION

Positive or plus-strand RNA viruses share many similarities in genomicorganization and structure, most notably a single-stranded coding RNA ofpositive polarity. Representative (+) strand RNA viruses include thepicornaviruses, flaviviruses, togaviruses, coronaviruses, andcaliciviruses. One clinically significant representative of theflavivirus family is the hepatitis C virus (HCV), the causative agentfor hepatitis C. Hepatitis C is an often chronic inflammatory disease ofthe liver which typically results in fibrosis and liver cancer (Choo, etal., Science (1989) 244:359). Infection by HCV typically results fromcontact with contaminated blood or blood products.

During HCV replication, a replicative (minus) RNA strand is producedwhich serves as a template for generation of several coding (+) RNAstrands. The HCV genome, which contains approximately 9600 nucleotides,is translated into a polyprotein consisting of approximately 3000 aminoacids (Leinbach, et al., Virology (1994) 204:163-169; Kato, et al., FEBSLetters (1991) 280:325-328). This polyprotein subsequently undergoespost-translational cleavage, producing several proteins. Due to highgenetic variability and mutation rates, the HCV comprises severaldistinct HCV genotypes that share approximately 70% sequence identity(Simmonds, et al., J. Gen. Virol., (1994) 75:1053-1061). Despite thishypervariability, there are three regions of the HCV genome that arehighly conserved, including the 5′- and 3′-non-coding regions, known asthe 5′-untranslated region (5′-UTR) and 3′-untranslated region (3′-UTR),respectively. These regions are thought to be vital for HCV RNAreplication as well as translation of the HCV polyprotein. In general,treatment of HCV is complicated by its high mutation rate, as well asthe mode of transmission and possibility of simultaneous infection withmultiple HCV genotypes.

Hepatitis C has several clinical phases. The first phase (i.e., acutephase) begins with infection by HCV. During this early phase, it ispossible to detect HCV-RNA in the serum of patients using polymerasechain reaction (PCR). However, because only about 25% of patientsexhibit jaundice during this phase, most cases (75%) go undetected inthe early stages. The inflammatory process, characterized by an increasein serum liver enzyme concentrations, begins approximately four weekspost infection. Although acute HCV infection is not malignant, themajority of patients (approximately 80%) develop chronic liver disease,characterized by a permanent elevation in the serum alanineaminotransferase level. Cirrhosis of the liver develops in more than 20%of patients with chronic HCV disease, which frequently leads tomalignant hepatoma. Life expectancy following diagnosis of the malignanthepatoma is generally 12 months.

Current therapies to treat HCV infections have met with limited success,with only a minority of patients experiencing long-term improvement. Themost prevalent treatment today involves specific cytokines known asinterferons, particularly interferon-α (IFN-alpha) which reduces serumalanine aminotransferase levels in approximately 50% of patients.Unfortunately, serum levels of alanine aminotransferase usually returnto elevated levels following termination of treatment, producing anumber of adverse side effects (Dusheiko, et al., J. Viral Hepatitis(1994) 1:3). Despite these problems, IFN-alpha is commonly used toreduce the risk of cirrhosis of the liver and malignant hepatoma. Thereis no currently available vaccine for HCV.

Although IFN-alpha remains the conventional approach, virologists haveevaluated a number of potential alternative therapies, including the useof specific ribozymes to inhibit translation of viral protein. Welch etal. disclose a two vector-expressed hairpin ribozyme directed againstHCV (Welch, et al., Gene Therapy (1996), 3(11):994). Lieber et al.report the removal of HCV-RNA in infected human hepatocytes throughadenovirus-mediated expression of specific hammerhead ribozymes (Lieber,et al., Virology (1996), 70 (12):8782). WO 99/55847 reports thedegradation of 5′- and 3′-UTL regions of HCV-RNA, as well as the5′-coding region for the nucleoprotein, using ribozymes. U.S. Pat. No.5,610,054 discloses enzymatic nucleic acid molecules that can inhibitreplication of HCV. Despite these efforts, the therapeutic value ofribozymes for treating HCV infections remains questionable, particularlyin view of their low enzymatic activity.

More recently, double-stranded RNA molecules (dsRNA) have been shown toblock gene expression in a highly conserved regulatory mechanism knownas RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNAinto small interfering RNAs (siRNA) of approximately 22 nucleotides,which serve as guide sequences to induce target-specific mRNA cleavageby an RNA-induced silencing complex RISC (Hammond, S. M., et al., Nature(2000), 404:293-296). When administered to a cell or organism, exogenousdsRNA has been shown to direct the sequence-specific degradation ofendogenous messenger RNA (mRNA) through RNAi. WO 99/32619 (Fires et al.)discloses the use of a dsRNA of at least 25 nucleotides in length toinhibit the expression of a target gene in C. elegans. dsRNA has alsobeen shown to degrade target RNA in other organisms, including plants(see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz etal.); Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000)10:1191-1200); and mammals (WO 00/44895, Limmer).

Despite significant advances in the field, there remains a need for anagent that can inhibit the replication of a virus in a host cell usingthe cell's own RNAi machinery. More specifically, an agent that has highbiological activity and can provide long-term, effective inhibition ofviral replication at a low dose would be highly desirable. Compositionscomprising such agents would be useful for treating a variety of viralinfections, including HCV.

SUMMARY OF THE INVENTION

The present invention discloses double-stranded ribonucleic acid(dsRNA), as well as compositions and methods for inhibiting thereplication of a (+) strand RNA virus, such as a Hepatitis C Virus(HCV). In particular, the invention relates to a dsRNA having an RNAstrand (the complementary strand) comprising a region which iscomplementary to at least a portion of a 3′-untranslated region (3′-UTR)of a (+) strand RNA virus. The present invention also disclosescompositions and methods for treating hepatitis C or HCV-associateddiseases.

In one aspect, the invention relates to a dsRNA. The dsRNA comprises asense RNA strand comprising a nucleotide sequence which is substantiallyidentical to at least a part of a 3′-untranslated region (3′-UTR) of a(+) strand RNA virus, and the dsRNA is less than 30 nucleotides inlength. The (+) strand RNA may be a hepatitis C virus. The dsRNA mayfurther comprise a complementary RNA strand, wherein the complementaryRNA strand comprises a complementary nucleotide sequence which is lessthan 30 nucleotides in length and is complementary to at least a portionof the 3′-UTR of the virus. In a preferred embodiment, the nucleotidesequence is within a highly conserved region of the 3′-UTR. Thecomplementary nucleotide sequence is preferably less than 25 nucleotidesin length, more preferably 21 to 24 nucleotides in length, and mostpreferably 23 nucleotides in length. The dsRNA may comprise one or twoblunt ends. The complementary RNA strand and the sense RNA strand maycomprise a 3′-terminus and a 5′-terminus, and at least one of the RNAstrands may comprise a nucleotide overhang of 1 to 3 nucleotides inlength, preferably two nucleotides in length. The dsRNA may furthercomprise two ends, wherein one end comprises the 3′-terminus of thecomplementary RNA strand and the 5′-terminus of the sense RNA strand,and the other end comprises the 5′-terminus of the complementary RNAstrand and the 3′-terminus of the sense RNA strand. In one embodiment,one end of the dsRNA end has a nucleotide overhang, preferably on the3′-terminus of the complementary RNA strand, and the second end isblunt. In another embodiment, the complementary RNA strand is 24nucleotides in length and the sense RNA strand is 22 nucleotides inlength, the 3′-end of the complementary RNA strand has a 2-nucleotideoverhang, and the other end of the dsRNA is blunt. In a particularembodiment, the complementary RNA strand comprises the nucleotidesequence of SEQ ID NO:5 and the sense RNA strand comprises thenucleotide sequence of SEQ ID NO:4.

In another aspect, the invention relates to a pharmaceutical compositionfor inhibiting the replication of a (+) strand RNA virus in an organism,such as a mammal (e.g., human). The pharmaceutical composition comprisesthe dsRNA as described above, together with a pharmaceuticallyacceptable carrier. The dosage unit of dsRNA in the composition may beless than 5 milligram (mg) of dsRNA per kg body weight, preferably 0.01to 2.5 milligrams (mg), more preferably 0.1 to 200 micrograms (μg), andmost preferably 0.1 to 100 μg per kilogram body weight. In oneembodiment, the pharmaceutically acceptable carrier is an aqueoussolution, such as phosphate buffered saline. In another embodiment, thepharmaceutically acceptable carrier comprises a micellar structure, sucha liposome, capsid, capsoid, polymeric nanocapsule, or polymericmicrocapsule. The pharmaceutical composition may be formulated to beadministered by inhalation, infusion, injection, or orally. In oneembodiment, the pharmaceutical composition is formulated to beadministered by intravenous or intraperitoneal injection.

In yet another aspect, the invention relates to a method for inhibitingthe replication of a (+) strand RNA virus comprising a 3′-untranslatedregion (3′-UTR) in a cell. The method comprises introducing adouble-stranded ribonucleic acid (dsRNA), as described above, into thecell. The dsRNA comprises a nucleotide sequence which is substantiallyidentical to at least a part of the 3′-UTR, and the dsRNA is less than30 nucleotides in length, more preferably less than 25 nucleotides, morepreferably 21 to 24 nucleotides, and most preferably 23 nucleotides inlength.

In still another aspect, the invention relates to a method for treatinga disease associated with infection of a (+) strand RNA virus in anorganism. The method comprises administering a pharmaceuticalcomposition to the organism, wherein the pharmaceutical compositioncomprises a double-stranded ribonucleic acid (dsRNA), as describedabove, together with a pharmaceutically acceptable carrier. The dsRNAcomprises a nucleotide sequence which is substantially identical to atleast a part of the 3′-UTR of the (+) strand RNA virus, and the dsRNA isless than 30 nucleotides in length.

The details of once or more embodiments of the invention are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the relevant sequence region from the p2 plasmid (SEQ ID NO2) and the N-terminal amino acid sequence of the corresponding reporterprotein (SEQ ID NO 2).

FIG. 2 shows the relevant sequence region from the p3 plasmid (SEQ ID NO3) and the N-terminal amino acid sequence of the corresponding reporterprotein (SEQ ID NO 3).

FIG. 3 shows the HCV1-2 dsRNA (SEQ ID NO 4, SEQ ID NO 5) in contrast tothe HCV sequence of an mRNA (SEQ ID NO 15) formed by means of the p2 andp3 plasmids.

FIG. 4 shows the GAL1-2 dsRNA (SEQ ID NO 6, SEQ ID NO 7) in contrast tothe mRNA sequence (SEQ ID NO 16) corresponding to β-gal gene (positivecontrol).

FIG. 5 shows the HCV3-4 the dsRNA (SEQ ID NO 8, SEQ ID NO 9), thatexhibits no relation to the expressed genes (negative control).

FIG. 6 shows the K22 dsRNA (SEQ ID NO 10, SEQ ID NO 11), that exhibitsno relation to the expressed genes (negative control).

FIG. 7 shows the antisense oligonucleotides HCVPT01 (SEQ ID NO 12),HCVPTO2 (SEQ ID NO 13), and HCVPTO3 (SEQ ID NO 14), in comparison to theHCV sequence of mRNA (SEQ ID NO 15) formed by the p3 plasmid.

FIG. 8 shows the effect of various concentrations of HCV1-2, GAL1-2, andHCV3-4 dsRNAs on the activity of β-galactosidase expressed by means ofthe p2 plasmid.

FIG. 9 shows the effect of various concentrations of HCV1-2, GAL-2, andHCV3-4 dsRNAs on the activity of β-galactosidase expressed by means ofthe p3 plasmid.

FIG. 10 shows the effect of the antisense oligonucleotides HCVPTO1,HCVPTO2, and HCVPTO3 of dsRNAs HCV1-2, GAL1-2, and HCV3-4 on theactivity of β-galactosidase expressed by means of the p3 plasmid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses double-stranded ribonucleic acid(dsRNA), as well as compositions and methods for inhibiting thereplication of a (+) strand RNA virus, such as a Hepatitis C Virus(HCV), using the dsRNA. The present invention also disclosescompositions and methods for treating diseases in organisms caused byinfection with HCV or HCV-associated diseases. dsRNA directs thesequence-specific degradation of mRNA through a process known as RNAinterference (RNAi). The process occurs in a wide variety of organisms,including mammals and other vertebrates. The dsRNA of the inventioncomprises an RNA strand (the complementary strand) having a region thatis complementary to at least a portion of a 3′-untranslated region(3′-UTR) of a (+) strand RNA virus. Using a cell-based assay, thepresent inventors have demonstrated that very low dosages of these dsRNAcan specifically and efficiently mediate RNAi in mammalian cells,resulting in a significant reduction in the activity or level of RNAencoded by the HCV genome as compared to untreated control cells. Thepresent invention encompasses these dsRNAs and compositions comprisingdsRNA and their use for specifically inhibiting the activity orreplication of a (+) strand RNA virus such as HCV. The use of thesedsRNAs enables the targeted degradation of mRNAs of genes that areimplicated in (+) RNA strand viral infections, including Hepatitis C.Thus, the methods and compositions of the present invention comprisingthese dsRNAs are useful for treating HCV and HCV-associated diseases.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the activity orreplication of a (+) strand RNA virus, as well as compositions andmethods for treating viral diseases. The pharmaceutical compositions ofthe present invention comprise a dsRNA having a complementary nucleotidesequence of less than 30 nucleotides in length, preferably less than 25nucleotides in length, and most preferably 21 to 24 nucleotides inlength, and which is substantially identical to at least a part of a3′-UTR of a (+) strand RNA virus, together with a pharmaceuticallyacceptable carrier. The dsRNA is less than 30 nucleotides in length,preferably less than 25 nucleotides in length, and most preferably 21 to24 nucleotides in length. The dsRNA may be blunt ended, or one end,preferably the 3′-end of the complementary (antisense) strand, may havea single-stranded nucleotide overhang of 1 to 3 nucleotides, preferably2 nucleotides in length.

Accordingly, certain aspects of the present invention relate topharmaceutical compositions comprising the dsRNA of the presentinvention together with a pharmaceutically acceptable carrier, methodsof using the compositions to inhibit the activity or replication of (+)strand RNA viruses such as HCV, and methods of using the pharmaceuticalcompositions to treat Hepatitis C and HCV-associated diseases.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

As used herein, the terms “3′-untranslated region” and “3′-UTR” refer tothe conserved, non-coding region at the 3′-end of a viral genome. The3′-UTR can be the entire non-coding region or a fragment thereof. Asused herein, the term “highly conserved region” refers to a region ofthe viral genome that remains evolutionarily constant, i.e., a genomicregion that has a very low mutation rate and thus shares significantsequence identity (>99%) between distinct viral genotypes.

The term “complementary RNA strand” (also referred to herein as the“antisense strand”) refers to the strand of a dsRNA which iscomplementary to a 3′-UTR of a (+) strand RNA virus. As used herein, theterm “complementary nucleotide sequence” refers to the region on thecomplementary RNA strand that is complementary to the 3′-UTR. “dsRNA”refers to a ribonucleic acid molecule having a duplex structurecomprising two complementary and anti-parallel nucleic acid strands. Notall nucleotides of a dsRNA must exhibit Watson-Crick base pairs; the twoRNA strands may be substantially complementary (i.e., having no morethan one or two nucleotide mismatches). The maximum number of base pairsis the number of nucleotides in the shortest strand of the dsRNA. TheRNA strands may have the same or a different number of nucleotides.Similarly, the complementary nucleotide sequence is less than 30,preferably less than 25, and most preferably 21 to 24 nucleotides inlength. The dsRNA is also preferably less than 30, more preferably lessthan 25, and most preferably 21 to 24 nucleotides in length. Thus, thelength of the dsRNA preferably corresponds to the length of thecomplementary nucleotide sequence. “Introducing into” means uptake orabsorption in the cell, as is understood by those skilled in the art.Absorption or uptake of dsRNA can occur through cellular processes, orby auxiliary agents or devices. For example, for in vivo delivery, dsRNAcan be injected into a tissue site or administered systemically. Invitro delivery includes methods known in the art such as electroporationand lipofection.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure when a3′-end of one RNA strand extends beyond the 5′-end of the othercomplementary strand, or vice versa. “Blunt” or “blunt end” means thatthe lengths of the two RNA strand are the same at that end of the dsRNA,and hence there is no nucleotide(s) protrusion (i.e., no nucleotideoverhang).

As used herein and as known in the art, the term “identity” is therelationship between two or more polynucleotide sequences, as determinedby comparing the sequences. Identity also means the degree of sequencerelatedness between polynucleotide sequences, as determined by the matchbetween strings of such sequences. Identity can be readily calculated(see, e.g., Computation Molecular Biology, Lesk, A. M., eds., OxfordUniversity Press, New York (1998), and Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York (1993),both of which are incorporated by reference herein). While there exist anumber of methods to measure identity between two polynucleotidesequences, the term is well known to skilled artisans (see, e.g.,Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J.,eds., M. Stockton Press, New York (1991)). Methods commonly employed todetermine identity between sequences include, for example, thosedisclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988)48:1073. “Substantially identical,” as used herein, means there is avery high degree of homology (preferably 100% sequence identity) betweenthe sense strand of the dsRNA and the corresponding part of the target3′-UTR of the viral genome. However, dsRNA having greater than 90%, or95% sequence identity may be used in the present invention, and thussequence variations that might be expected due to genetic mutation,strain polymorphism, or evolutionary divergence can be tolerated.Although 100% identity is preferred, the dsRNA may contain single ormultiple base-pair random mismatches between the RNA and the target3′-UTR.

As used herein, the term “treatment” refers to the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder, e.g., a disease or condition, asymptom of disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier ordiluent for administration of a therapeutic agent. Pharmaceuticallyacceptable carriers for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed. 1985),which is hereby incorporated by reference herein. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The term specificallyexcludes cell culture medium. For drugs administered orally,pharmaceutically acceptable carriers include, but are not limited topharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a dsRNAmolecule has been introduced by means of recombinant DNA techniques.

II. Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention relates to a double-strandedribonucleic acid (dsRNA) having a nucleotide sequence which issubstantially identical to at least a portion of a target 3′-UTR of a(+) strand RNA virus. The dsRNA comprises two RNA strands that aresufficiently complementary to hybridize to form the duplex structure.

One strand of the dsRNA comprises the nucleotide sequence that issubstantially identical to a portion of the target 3′-UTR (the “sense”strand), and the other strand (the “complementary” or “antisense”strand) comprises a sequence that is complementary to the 3′-UTR.Because of this complementarity, the complementary RNA strand is able tobase-pair with the complementary region of the 3′-UTR, thus inducing astructural change within the target 3′-UTR. For example, thecomplementary region of the 3′-UTR may be cleaved (through RNAinterference) and/or ligated to other nucleic acid molecules, thusresulting in degradation and/or insertion or deletion mutations. Bindingbetween the complementary RNA strand and the target 3′-UTR can alsoinduce a structural change in the secondary and/or tertiary structure ofthe 3′-UTR. Because this region is vital for viral replication, suchstructural changes can block or significantly inhibit replication.Moreover, due to the high sequence variability of the genome of (+)strand RNA viruses, particularly HCV, sdRNAs that target conservedregions of the 3′UTR may have a significant impact over a wide range ofviral genotypes. Thus, not only is the efficiency of inhibition of viralreplication increased by targeting a highly conserved region of the3′-UTR, but targeting such regions also enables the treatment of diversepatient populations.

The sequence that is complementary to the target 3′-UTR (i.e., thecomplementary nucleotide sequence) is less than 30 nucleotides,preferably less than 25 nucleotides, and most preferably 21 to 24nucleotides in length. Similarly, the dsRNA may have less than 30nucleotides, preferably less than 25 nucleotides, and most preferably 21to 24 nucleotides in length. The dsRNA can be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer,such as are commercially available from Biosearch, Applied Biosystems,Inc. In specific embodiments, the dsRNA can comprise the sequence setforth in SEQ ID NOS:12 or 13, or a complement thereof. In a particularembodiment, the antisense (complementary) RNA strand comprises thesequence set forth in SEQ ID NO:5, and the sense RNA strand comprisesthe sequence set forth in SEQ ID NO:4.

In one embodiment, at least one end of the dsRNA is blunt. dsRNA with atleast one blunt end show improved stability as compared to dsRNA havingtwo nucleotide overhangs. dsRNA with at least one blunt end showsgreater in vivo stability (i.e., is more resistant to degradation in theblood, plasma, and cells). However, dsRNAs having at least onenucleotide overhang have unexpectedly superior inhibitory propertiesthan their blunt-ended counterparts. Moreover, the present inventorshave discovered that the presence of only one nucleotide overhangstrengthens the interference activity of the dsRNA, without affectingits overall stability. The stability, particularly plasma stability, canthus be adjusted in accordance with needs of the particular application.dsRNA having only one overhang has proven particularly effective in vivo(as well as in a variety of cells, and cell culture mediums), and aremore stable than dsRNA having two blunt ends. The single-strandednucleotide overhang may be 1 to 3, preferably two, nucleotides inlength. Preferably, the single-stranded overhang is located at the3′-end of the complementary (antisense) RNA strand. Such dsRNAs haveimproved stability and inhibitory activity, thus allowing administrationat low dosages, i.e., less than 5 mg/kg body weight of the recipient perday. Preferably, the complementary strand of the dsRNA has a nucleotideoverhang at the 3′-end, and the 5′-end is blunt.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a dsRNA, as described in the preceding section, and apharmaceutically acceptable carrier, as described below. Thepharmaceutical composition comprising the dsRNA is useful for treatingan infection or disease associated with the activity or replication of a(+) strand RNA virus.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit the activity orreplication of a (+) strand RNA virus, such as HCV. The presentinventors have found that compositions comprising the dsRNA of theinvention can be administered at surprisingly low dosages. A maximumdosage of 5 mg dsRNA per kilogram body weight per day is sufficient toinhibit or completely suppress the activity or replication of the targetvirus.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, preferablyin the range of 0.1 to 2.5 milligrams per kilogram body weight of therecipient per day, more preferably in the range of 0.1 to 200 microgramsper kilogram body weight per day, and most preferably in the range of0.1 to 100 micrograms per kilogram body weight per day. Thepharmaceutical composition may be administered once daily, or the dsRNAmay be administered as two, three, four, five, six or more sub-doses atappropriate intervals throughout the day. In that case, the dsRNAcontained in each sub-dose must be correspondingly smaller in order toachieve the total daily dosage. The dosage unit can also be compoundedfor delivery over several days, e.g., using a conventional sustainedrelease formulation which provides sustained release of the dsRNA over aseveral day period. Sustained release formulations are well known in theart. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the infection or disease, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse repositories canbe found at The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of dsRNA, as well as for determining a therapeuticallyeffective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredby intravenous or intraparenteral infusion or injection.

For oral administration, the dsRNAs useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of dsRNA inthe cells that harbor the virus. Such substances include, for example,micellar structures, such as liposomes or capsids, as described below.Surprisingly, the present inventors have discovered that compositionscontaining only naked dsRNA and a physiologically acceptable solvent aretaken up by cells, where the dsRNA effectively inhibits replication ofthe virus. Although microinjection, lipofection, viruses, viroids,capsids, capsoids, or other auxiliary agents are required to introducedsRNA into cell cultures, surprisingly these methods and agents are notnecessary for uptake of dsRNA in vivo. Aqueous suspensions according tothe invention may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydxoxybenzoate.

The pharmaceutical compositions useful according to the invention alsoinclude encapsulated formulations to protect the dsRNA against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075, which areincorporated by reference herein.

Toxicity and therapeutic efficacy of dsRNAs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD5O/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs useful according to the invention can beadministered in combination with other known agents effective intreating viral infections and diseases. In any event, the administeringphysician can adjust the amount and timing of dsRNA administration onthe basis of results observed using standard measures of efficacy knownin the art or described herein.

For oral administration, the dsRNAs useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

IV. Methods for Treating Viral Infections and Diseases

In one embodiment, the invention relates to a method for treating asubject having an infection or a disease associated with the replicationor activity of a (+) strand RNA virus having a 3′-UTR, such as HCV. Inthis embodiment, the dsRNA can act as novel therapeutic agents forinhibiting replication of the virus. The method comprises administeringa pharmaceutical composition of the invention to the patient (e.g.,human), such that viral replication is inhibited. Because of their highspecificity, the dsRNAs of the present invention specifically target (+)strand RNA viruses having a 3′-UTR, as described above, and atsurprisingly low dosages.

Examples of (+) strand RNA viruses which can be targeted for inhibitioninclude, without limitation, picornaviruses, caliciviruses, nodaviruses,coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples ofpicornaviruses include enterovirus (poliovirus 1), rhinovirus (humanrhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus(encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virusO), and parechovirus (human echovirus 22). Examples of calicivirusesinclude vesiculovirus (swine vesicular exanthema virus), lagovirus(rabbit hemorrhagic disease virus), “Norwalk-like viruses” (Norwalkvirus), “Sapporo-like viruses” (Sapporo virus), and “hepatitis E-likeviruses” (hepatitis E virus). Betanodavirus (striped jack nervousnecrosis virus) is the representative nodavirus. Coronaviruses includecoronavirus (avian infections bronchitis virus) and torovirus (Bernevirus). Arterivirus (equine arteritis virus) is the representativearteriviridus. Togaviruses include alphavirus (Sindbis virus) andrubivirus (Rubella virus). Finally, the flaviviruses include flavivirus(Yellow fever virus), pestivirus (bovine diarrhea virus), andhepacivirus (hepatitis C virus). In a preferred embodiment, the virus ishepacivirus, the hepatitis C virus. Although the foregoing listexemplifies vertebrate viruses, the present invention encompasses thecompositions and methods for treating infections and diseases caused byany (+) strand RNA virus having a 3′-UTR, regardless of the host. Forexample, the invention encompasses the treatment of plant diseasescaused by sequiviruses, comoviruses, potyviruses, sobemovirus,luteoviruses, tombusviruses, tobavirus, tobravirus, bromoviruses, andclosteroviruses.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredby intravenous or intraparenteral infusion or injection.

V. Methods for Inhibiting Expression of a Target Gene

In yet another aspect, the invention relates to a method for inhibitingthe replication or activity of a (+) strand RNA virus, such as HCV. Themethod comprises administering a composition of the invention to thehost organism such that replication of the target virus is inhibited.The organism may be an animal or a plant. Because of their highspecificity, the dsRNAs of the present invention specifically target (+)strand RNA viruses having a 3′-UTR, and at surprisingly low dosages.Compositions and methods for inhibiting the replication of a targetvirus using dsRNAs can be performed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of a 3′-UTR of a (+) strandRNA virus. When the organism to be treated is a mammal, such as a human,the composition may be administered by any means known in the artincluding, but not limited to oral or parenteral routes, includingintravenous, intramuscular, intraperitoneal, subcutaneous, transdermal,airway (aerosol), rectal, vaginal and topical (including buccal andsublingual) administration. In preferred embodiments, the compositionsare administered by intravenous or intraparenteral infusion orinjection.

The methods for inhibiting viral replication can be applied to any (+)strand RNA virus, such as those described above.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES Example 1 Inhibition of the 3′-UTR of HCV

To study RNA interference and the action of antisense oligonucleotidesin a non-pathogenic assay, Sequence No. 1 in the sequence protocol wascloned in front of a gene that codes for E. coli β-galactosidase.Sequence No. 1 corresponds to a sequence from a highly conserved regionof the 3′-UTR of the HCV genome that is 24 nucleotides in length. Aftertransfection of the 3′-UTR plasmid in human HuH-7 liver cells, thesequence was transcribed as a part of an mRNA that codes for13-galactosidase. The mRNA sequence that corresponds to the 3′UTR istherefore identical to the HCV genome sequence and was subsequently usedas the target sequence.

Generation of p2 and p3 Reporter Plasmids

The E. coli β-galactosidase (β-gal) gene was isolated from thecommercially available expression vector pβ-Gal control (BD BiosciencesClontech, Tullastr. 4, 69126 Heidelberg, Germany, Gene Accession No.U13186, Nucleotide 280-3429).

The HCV sequence is part of a fusion gene in the p2 plasmid. The HCVsequence is part of the open reading frame of the sequence that codesfor β-galactosidase, so that the HCV sequence is also expressed as partof a fusion protein. FIG. 1 shows the relevant sequence segments of thep2 plasmid (Sequence No. 2 of the sequence protocol). The HCV sequenceis shown in italics. The beginning of the β-Gal gene (including 6nucleotides of the Kozak sequence in front of the ATG codon) isunderlined. The N-terminal amino acid sequence of the HCVβ-galactosidase fusion protein is listed under the DNA sequence.

The HCV sequence is also part of a fusion gene in the p3 plasmid.However, the HCV sequence is located outside of the open reading frameof the sequence that codes for β-galactosidase, so that the HCV sequenceis not expressed as part of a fusion protein. FIG. 2 shows the relevantsequence segment of the p3 plasmid (Sequence No. 3 in the sequenceprotocol). The HCV sequence is shown in italics. The beginning of theβ-Gal gene (including 6 nucleotides of the Kozak sequence in front ofthe ATG codon) is underlined. The N-terminal amino acid sequence of theexpressed β-galactosidase is listed under the DNA sequence.

The fusion genes that were generated in this way were cloned into thecommercially available pcDNA3.1 (+) expression plasmid (Invitrogen, LifeTechnologies, Karlsruhe Technology Part, Emmy Noether Str. 10, 76131Karlsruhe, Germany; Catalogue No. V790-20). This plasmid contains aneomycin resistance gene and thus confers on the HuH-7 cells that aretransfected with it resistance to the G418. HuH-7 cells selected in thepresence of G418 therefore harbor a reporter plasmid that stablyintegrated into the cell's genome. The commercially available pGL3-ctrlplasmid (Promega GmbH, High Tech Park, Schildkrötstr. 15, 68199Mannheim, Germany; Gene Accession No. U47296 was used as the controlplasmid. It codes and expresses the “firefly luciferase” gene.

Synthesis and Preparation of dsRNAs

Oligoribonucleotides are synthesized with an RNA synthesizer (Expedite8909, Applied Biosystems, Weiterstadt, Germany) and purified by HighPressure Liquid Chromatography (HPLC) using NucleoPac PA-100 columns,9×250 mm (Dionex Corp.; low salt buffer: 20 mM Tris, 10 mM NaClO₄, pH6.8, 10% acetonitrile; the high-salt buffer was: 20 mM Tris, 400 mMNaClO4, pH 6.8, 10% acetonitrile. flow rate: 3 ml/min). Formation ofdouble stranded dsRNAs is then achieved by heating a stoichiometricmixture of the individual complementary strands (10 M) in 10 mM sodiumphosphate buffer, pH 6.8, 100 mM NaCl, to 80-90° C., with subsequentslow cooling to room temperature over 6 hours.

In addition, dsRNA molecules with linkers may be produced by solid phasesynthesis and addition of hexaethylene glycol as a non-nucleotide linker(D. Jeremy Williams, Kathleen B. Hall, Biochem (1996) 35:14665-14670). AHexaethylene glycol linker phosphoramidite (Chruachem Ltd, Todd Campus,West of Scotland Science Park, Acre Road, Glasgow, G20 OUA, Scotland,UK) is coupled to the support bound oligoribonucleotide employing thesame synthetic cycle as for standard nucleoside phosphoramidites(Proligo Biochemie GmbH, Georg-Hyken-Str.14, Hamburg, Germany) but withprolonged coupling times. Incorporation of linker phosphoramidite iscomparable to the incorporation of nucleoside phosphoramidites.

DsRNA Oligonucleotides

Three short double-stranded ribonucleic acids (dsRNA) were used for theRNA interference. These dsRNAs each consist of 2 shortoligoribonucleotides that are complementary to each other over almostthe entire sequence region. Two nucleotides have no base pairing ateither of the 3′-ends of the oligoribonucleotides, and therefore formdsRNA overhangs.

The sequence of one of the oligoribonucleotides is identical to the mRNAtarget sequence. This oligoribonucleotide is therefore called the sensestrand. The sequence of the other oligoribonucleotide is complementaryto the mRNA target sequence. This oligoribonucleotide is thereforecalled the antisense strand.

The double-stranded oligoribonucleotide designated as HCV1-2 is shown inFIG. 3 and compared to the HCV sequence of the mRNA formed by means ofthe p2 and p3 plasmids. The nucleotides shown in capital letterscorrespond to the HCV sequence in the p2 and p3 plasmids. HCV1-2consists of the HCV 1 sense strand and the HCV 2 antisense strand,whereby two nucleotides in each exhibit no base pairing at the 3′-endsof the strands. The sense strand (HCV 1) depicted in Sequence No. 4 inthe sequence protocol exhibits almost the same nucleotide sequence asthe HCV sequence of an mRNA formed by means of the p2 and p3 plasmids,respectively. Three nucleotides of the HCV sequence are missing at the5′-end, and two nucleotides are present at the 3′-end that are not acomponent of the HCV sequence. The antisense strand (HCV 2) depicted inSequence No. 5 in the sequence protocol is, except for the twonucleotides at the 3′-end, complementary to HCV 1, and therefore also tothe HCV sequence of an mRNA formed by means of the p2 and p3 plasmids,respectively. The HCV sequence corresponds to a 3′-untranslated regionof the HCV genome.

A dsRNA designated as GAL1-2 was used as the positive control. It isshown in FIG. 4 in contrast to an mRNA sequence (designated as mRNA inFIG. 4) that corresponds to the β-Gal gene of the p2 and p3 plasmids.GAL1-2 consists of the Gal 1 sense strand and the Gal 2 antisensestrand, whereas two nucleotides in each exhibit no base pairing at the3′-ends of the strands. The sense strand (Gal 1) shown in Sequence No. 6in the sequence protocol exhibits almost the same nucleotide sequence asthe mRNA sequence that corresponds to the β-Gal gene. The antisensestrand (Gal 2) shown in Sequence No. 7 in the sequence protocol is,except for the two nucleotides at the 3′-end, complementary to Gal 1,and therefore also to the mRNA sequence that corresponds to the β-Galgene.

In one part of the experiment, a dsRNA designated as HCV3-4, which hasno relationship to the genes expressed here, was used as the negativecontrol (FIG. 5). HCV3-4 consists of the HCV 3 sense strand and the HCV4 antisense strand, whereby two nucleotides in each exhibit no basepairing at the 3′-ends of the strands. The sense strand (HCV 3) shown inSequence No. 8 of the sequence protocol exhibits almost no similarity tothe mRNA formed by means of the p2 and p3 plasmids, and therefore has norelationship to the expressed genes. The antisense strand (HCV 4) shownin Sequence No. 9 in the sequence protocol is, except for the twonucleotides at the 3′-end, complementary to HCV 3 and therefore also hasno relationship to the mRNA that is formed.

In another part of the experiment, a dsRNA designated as K22 was used asthe negative control. It also exhibits no relationship to the geneexpressed here (FIG. 6). The sequences of both oligoribonucleotides thatform the dsRNA are shown in Sequence Nos. 10 and 11 in the sequenceprotocol.

Three 21-nucleotide-long DNA antisense oligoribonucleotides were used asphosphothioates in the experiments on antisense oligoribonucleotides.The oligoribonucleotides were obtained from Metabion GmbH, Lena-ChristStr. 44, 82152 Martinsried, Germany. They are here designated asHCVPTOI, HCVPTO2, and HCVPTO3. HCVPTO1 and HCVPTO2 are complementary todifferent regions of the HCV-mRNA sequence formed by means of the p3plasmid. HCVPTO3 is the negative control without relationship to thetarget sequence. HCVPTO1, HCVPTO2, and HCVPTO3 are shown in FIG. 7 incontrast to the HCV-mRNA sequence. RNA interference assays were testedon the HuH-7 type liver cell line (Nakabayashi et al. 1982 which can beinfected by HCV and is used routinely to culture these viruses. Thecells were cultured in DMEM (Dulbecco's Modified Eagle Medium) with 10%fetal calf serum (FCS).

a) Experiments Relating to RNA Interference Transfection

Prior to transfection, 2×10⁴ cells per well of a 96-well cell cultureplate were seeded. 3 μg p2 plasmid and p3 plasmid, respectively, weremixed with 1 μg pGL3-ctrl plasmid. 0.25 μg of this plasmid mixture wasplaced in each well for transfection. Approximately 24 hours afterseeding the cells, the p2/pGL3-ctrl and p3/pGL3-ctrl reporter plasmidswere transfected together with dsRNA in HuH-7. The quantity oftransfected DNA per well was constant.

The dsRNA was added to the plasmid mixtures in decreasing concentrationsof 400 nmol/l to 12.5 nmol/l (in relation to 110 μl total transfectionvolume). The initial concentration of the HCV1-2, GALL-2, andnonspecific HCV3-4 dsRNAs in each stock. solution was 20 μmol/l. ThedsRNAs were diluted by mixing them stepwise with the same volume ofannealing buffer (AB, 100 mmol/lNaCl, 20 mmol/l sodium phosphate, pH6.8) to arrive at the end concentration.

For an end concentration of 400 nmol/l, 2.2 μl stock solution was usedfor a transfection volume of 110 μl per well, and 6.6 μl stock solutionwas used for a transfection volume of 330 μl per well, respectively. Thedilution steps were produced as shown in Table 1.

TABLE 1 Production of dsRNA dilution steps Concentration of InitialQuantity of Quantity of End Solution Solution Initial Added ABConcentration* No. Initial Solution (μmol/l) Solution (μl) (μl) (nmol/l)1 Stock solution 20 14.0 400 2 Solution 1 10 7.0 7.0 200 3 Solution 2 57.0 7.0 100 4 Solution 3 2.5 7.0 7.0 50 5 Solution 4 1.25 7.0 7.0 25 6Solution 5 0.62 7.0 7.0 12.5 *End concentration, using 6.6 μl of eachsolution to a transfection volume of 330 μl.

Plasmids and dsRNA were cotransfected. Gene Porter 2 (PeQLab, CarlThiersch Str. 2B, 91052 Erlangen, Germany; Catalogue No. 13-T202007) wasused as the transfection agent. Each cotransfection was repeated threetimes.

For 3 wells of the 96-well plates a mixture was made that consisted of2.0 μl of a plasma mixture consisting of the p2 plasmid and the pGL3control plasmid (0.3875 μg/μl; 3:1), 6.6 μl dsRNA (20, 10, 5, 2.5, 12.5,and 0.62 μmol/l, respectively), and 16.4 μl DNA diluent B (suppliedtogether with Gene Porter 2, PeQLab). This mixture was mixed with amixture consisting of 6.0 μl Gene Porter 2 and 19 μl serum-free medium.The total volume of the resultant mixture was 50 μl, of which 16.5 μlwas added to each of 2×10⁴ HuH-7 in 100 μl of medium. Then a mixture wasmade that consisted of 2.0 μl of a plasmid mixture consisting of the p3plasmid and the pGL3 control plasmid (0.3875 μg/μl); 3:1), 6.6 μl dsRNA(20, 10, 5, 2.5, 12.5, and 0.62 μmol/l respectively), and 16.4 μl DNAdiluent B. This mixture was mixed with a mixture consisting of 6.0 μlGene Porter 2 and 19 μl serum-free medium. The total volume of theresultant mixture was 50 μl, of which 16.5 μl was added to each of 2×10⁴HuH-7 in 100 μl of medium. The transfected cells were incubated at 37°C. and 5% CO2. 35 μl of fresh medium was added to each well, and thecells were incubated for another 24 hours. The cells were thentrypsinied.

Detection Methods Used

The effect of dsRNA on the expression of the reporter genes wasdetermined by quantifying the μl-galactosidase and luciferase activityby means of chemoluminescence. For this, lysates were made using theTropix Lysebuffer (Applied Biosystems, 850 Lincoln Centre Drive, FosterCity, Calif. 944404; Catalogue No. BD100LP) in accordance withmanufacturer's instructions.

To quantify β-galactosidase activity, 2 p.1 lysate was used peranalysis, as well as the substrate Galacto Star (Applied Biosystems,Tropix; Catalogue No. BM100S), in accordance with manufacturerinstructions. To quantify luciferase activity, 5 gl lysate was used peranalysis, as well as the substrate Luciferin (Applied Biosystems,Tropix; Catalogue No. BM100L) in accordance with manufacturerinstructions. Luminescence was measured in each case using the BertholdSirius luminometer (Berthold Detection Systems GmbH, Bleichstr. 56-58,75173 Pforzheim, Germany).

Results

For each transfection assay, three 96-well plates were analyzed, suchthat in each case both β-galactosidase and luciferase were measured. Thequotient of the relative light units (RLU) of β-galactosidase and therelative light units of luciferase were calculated. An average wasdetermined for these three values. The average for p2/pGL3- and p3/pGL3transfected cells without dsRNA, respectively, was arbitrarily definedas 1.0. The values that changed under the influence of dsRNA wererecorded as a ratio to 1.0 (see FIGS. 8 and 9), i.e., a value of 0.6corresponds to a 40% inhibition of β-galactosidase activity incomparison with untreated cells. In FIG. 8, cotransfection ofsequence-specific dsRNA with the p2 plasmid resulted in a reduction inβ-galactosidase activity. The HCV1-2 and GALL-2 dsRNAs inhibitβ-galactosidase with comparable effectiveness. At transfection volumesof 400 mnol/l and 200 niol/l of dsRNA, β-galactosidase activitydecreases to 40% as compared to untreated cells. The inhibitory effectdecreased with decreasing dsRNA concentration. The HCV3-4 control dsRNAleads to no decrease in β-galactosidase activity in lysate over theentire concentration range. A reduction in f3-galactosidase expressionis also detectable with cotransfection of the sequence-specific HCV1-2dsRNA with the p3 plasmid (FIG. 9). HCV1-2 and GAL1-2 inhibitβ-galactosidase activity with comparable effectiveness. At transfectionvolumes of 400=Oland 200 mol/l of dsRNA, β-galactosidase activitydecreases to approximately 20% as compared to untreated cells. Theinhibitory effect decreased with decreasing dsRNA concentration. TheHCV3-4 control dsRNA showed a weak inhibition of β-galactosidaseactivity to approximately 70% as compared to untreated cells. In thepresence of the HCV1-2 dsRNA, both the p2 and p3 plasmids showed amarked decrease in β-galactosidase activity. Comparable effects wereseen with the GAL1-2 dsRNA (positive control). The second control dsRNA,HCV3-4, led to no and markedly less inhibition of β-galactosidaseactivity, respectively. Expression and/or stability of RNA was markedlydecreased by dsRNA in the experiments described. This was also true forHCV target sequences outside the open reading frame, which correspondsto the situation with the natural 3′-UTR region of HCV.

b) Experiments with Antisense DNA Oligonucleotides

To prepare for the experiments, p3 was stably transfected into HuH-7cells using LipofectaminePLUS (GIBCO BRL Life Technologies, KarlsruheTechnology Park, Emmy Noether Str. 10, 76131 Karlsruhe, Germany). Forthis, 2×10⁴ cells were seeded per well of a 96-well cell culture plate.After 24 hours, the medium was replaced with 50 μl serum-free medium(DMEM). The transfection mixture consisted of 0.2 μg p3, 16.7 μl DMEM, 2μl PLUS reagent, and 1 μl Lipofectamine reagent. Cells were transfectedin accordance with manufacturer's instructions. After three hours, thetransfection medium was replaced with 150 μl complete medium (DMEM+10%fetal calf serum). After 48 hours, the cells were transferred to wellsin a 12-well cell culture plate, and cultured with 400 μg/ml G418(Amersham Biosciences, Munzinger Str. 9, 79111 Freiberg, Germany).Colonies were collected and transferred to new wells in a 12-well cellculture plate. From these, the cells that grew in the new wells after14-21 days were culled manually and cultured with 400 μg/ml G418 untilthe selection was complete. After approximately three manual selections,β-galactosidase activity was determined as described below by means ofenzyme measurements. Then the number of cells that expressedgalactosidase was determined using X-Gal staining. For this, the mediumwas aspirated and the cells were stained in the wells of a 96-well cellculture plate overnight in 100 μl X-Gal solution (10 mmol/l sodiumphosphate, pH 7.0; 1 mmol/lMgCL₂; 150 mmol/l NaCl; 3.3mmol/lK₄Fe(CN)₆*3H₂0; 3.3 mmol/lK₄Fe(CN)₆; 0.2% X-Gal) (X-Gal fromPeQLab, Erlangen, Germany; all other chemicals from SIGMA, GriinwalcierWeg 30, 82024 Taufldrchen, Germany). The best clone was designated“HuH-7 blue” and used for the experiments.

Transfection with dsRNA and Antisense DNA Oligonucleotides

To prepare for a transfection, 2×10⁴ cells of HuH-7 blue was seeded in100 μl DMEM+10% FCS per well of a 96-well cell culture plate. After 24hours, the dsRNA and the antisense DNA oligonucleotides weretransfected. Fugene 6 (Roche Applied Sciences, Sandhofer Str. 116, 68305Mannheim, Germany; Catalogue No. 1814443) was used for thesetransfections. Every fifth well containing HuH-7 blue cells was nottreated. Stock solutions with a concentration of 20 μmol/l were madefrom the HCV 1-2, GAL1-2, and K22 dsRNAs. 1.6 μl of this stock solutionwas in each case mixed with 0.9 μl Fugene 6 and 108 μl DMEM. The dsRNAwas therefore present at a concentration of 15 nmol/l. Each of 5 wellsof a 96-well cell culture plate was transfected with 20 μl of thisassay. Stock solutions were made with each of the antisense DNAoligonucleotides HCVPTO1, HCVPTO2, and HCVPTO3, and a concentration of100 μmol/l. 1.2 μl of this stock solution was in each case mixed with2.4 μl Fugene 6 and 108 μl DMEM. The dsRNA was therefore present in aconcentration of 200 nmol/l. Each of 5 wells of a 96-well cell cultureplate was transfected with 20 μl of this mixture.

Detection Methods

The effect of dsRNA oligonucleotides and antisense DNA oligonucleotideson the expression of reporter genes was determined by quantifying theβ-galactosidase activity by means of chemoluminescence. For this,lysates were made using the Tropix Lysebuffer (Applied Biosystems, 850Lincoln Centre Drive, Foster City, Calif. 944404; Catalogue No. BD100LP)in accordance with manufacturer's instructions. Chemoluminescencemeasurements were quantified as follows:

5 μl of lysate were placed in each reagent vessel and filled to 30 μlwith β-Gal assay buffer (1 ml 1 mol/l sodium phosphate buffer, pH 8.0;10 μl 1 mol/l MgCl₂, 10 μl 1.25 mg/ml Galakton [Tropix GCO20, AppliedBiosystems]; 9 ml deiodized water). MI β-Gal stop mix (1 ml 2 mol/INa0H,250 μl 2.5% Emerald Enhancer [Applied Biosystems, Tropix, LAY250], 8.75ml deionized water), mixed thoroughly, and immediately measured in theluminometer. If not otherwise noted, all reagents were supplied bySIGMA. Luminescence was measured in each case using the Berthold Siriusluminometer (Berthold Detection Systems GmbH, Bleichstr. 56-58, 75173Pforzheim, Germany). 5 wells of a 96-well cell culture plate wereanalyzed per transfection assay. β-galactosidase activity was determinedin each case, and the average of the 5 individual values wasestablished. The average value for untransfected cells is arbitrarilydefined as 1.0. The average values for transfected cells are thenexpressed as a ratio with the average for untransfected cells. Forexample, a value of 0.6 corresponds to an inhibition of P-galactosidaseactivity by 40% in comparison to untreated cells. The results are shownin FIG. 10.

Results

With transfection of sequence-specific antisense oligonucleotides (200nmol/l) and dsRNA oligonucleotides (50 nmol/l) in the HuH-7 blue cellline, a reduction in β-galactosidase activity was detectable. HCVPTO1reduced the activity of β-galactosidase by 35%, and HCVPTO2 by 40%. TheHCVPTO3 oligonucleotide used as the negative control increased theactivity by 40% as compared to untreated cells. The HCV1-2 and GAL1-2dsRNAs inhibited β-galactosidase activity with comparable effectiveness.β-galactosidase activity decreased by 37% in each case, as compared withuntreated cells. The K22 nonspecific control increased activity by 15%in comparison with untreated cells.

Example 2 Treatment of a HCV Infected Patient with dsRNA

In this Example, HCV specific double stranded dsRNAs are injected intoHCV infected patients and shown to specifically inhibit HCV geneexpression.

dsRNA Administration and Dosage

The present example provides for pharmaceutical compositions for thetreatment of human HCV infected patients comprising a therapeuticallyeffective amount of a HCV specific dsRNA as disclosed herein, incombination with a pharmaceutically acceptable carrier or excipient.DsRNAs useful according to the invention may be formulated for oral orparenteral administration. The pharmaceutical compositions may beadministered in any effective, convenient manner including, forinstance, administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes, among others. One of skill in the art can readily prepare dsRNAsfor injection using such carriers that include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. Additional examples of suitable carriers are foundin standard pharmaceutical texts, e.g. “Remington's PharmaceuticalSciences”, 16th edition, Mack Publishing Company, Easton, Pa., 1980.

Example 3 RNA Purification and Analysis

Efficacy of the dsRNA treatment is determined at defined intervals afterthe initiation of treatment using real time PCR on total RNA extractedfrom peripheral blood. Cytoplasmic RNA from whole blood, taken prior toand during treatment, is purified with the help of the RNeasy Kit(Qiagen, Hilden) and HCV mRNA levels are quantitated by real time RT-PCRas described previously (Eder, M., et al., Leukemia (1999) 13:13831389;Scherr M et al., BioTechniques. (2001) 31:520-526).

Example 4 HCV-Specific dsRNA Expression Vectors

HCV-specific dsRNA molecules that interact with HCV target RNA moleculesand modulate HCV gene expression activity are expressed fromtranscription units inserted into DNA or RNA vectors (see, for example,Couture et A, 1996, TIG., 12, 5 1 0, Skillem et A, International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann etal., 1995, Proc. Natl. Acad. Sci. USA 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and cotransfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, the dsRNA is expressed as an inverted repeatjoined by a linker polynucleotide sequence such that the dsRNA has astem and loop structure.

The recombinant dsRNA expression vectors are preferably DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka et al. (1992, Curr. Topics in Micro. and Immunol. 158:97-129)),adenovirus (see, for example, Berkner et al. (1988, BioTechniques6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992, Cell 68:143-155)), or alphaviras as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see for example Eglitis, et al., 1985, Science230:1395-1398; Danos and Mulligan, 1988, Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or preferably RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from all promoter.The promoter can also direct transgene expression to the liver e.g.albumin regulatory sequence (Pinkert et al., 1987, Genes Dev. 1:268276).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Preferably, recombinant vectors capable of expressing dsRNA moleculesare delivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allow for introduction into a desired target cell.

DsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single target gene or multiple target genes over a period of a weekor more are also contemplated by the present invention. Successfulintroduction of the vectors of the invention into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofex vivo cells can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

The nucleic acid molecules of the invention described above can also begenerally inserted into vectors and used as gene therapy vectors forhuman patients infected with HCV. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting thereplication of a (+) strand RNA virus, wherein the dsRNA comprises asense RNA strand consisting of a nucleotide sequence which issubstantially identical to at least a part of a 3′-untranslated region(3′-UTR) of the (+) strand RNA virus and an antisense RNA strand whichis complementary to at least a part of the sense RNA strand, and whereineach strand of the dsRNA is 21 to 24 nucleotides in length. 2-3.(canceled)
 4. The dsRNA of claim 1, wherein the dsRNA comprises a bluntend.
 5. The dsRNA of claim 1, wherein the dsRNA comprises two bluntends.
 6. The dsRNA of claim 1, wherein the antisense RNA strand and thesense RNA strand comprise a 3′-terminus and a 5′-terminus, and whereinat least one of said RNA strands comprises a nucleotide overhang of 1 to3 nucleotides in length.
 7. The dsRNA of claim 6, wherein the nucleotideoverhang is two nucleotides in length.
 8. The dsRNA of claim 6, whereinthe nucleotide overhang is on the 3′-terminus of the antisense RNAstrand.
 9. The dsRNA of claim 6, wherein the dsRNA further comprises afirst end and a second end, wherein the first end comprises the3′-terminus of the antisense RNA strand and the 5′-terminus of the senseRNA strand, and wherein the second end comprises the 5′-terminus of theantisense RNA strand and the 3′-terminus of the sense RNA strand,wherein the first end comprises a nucleotide overhang on the 3′-terminusof the antisense RNA strand, and wherein the second end is blunt. 10.The dsRNA of claim 6, wherein the antisense RNA strand is 23 nucleotidesin length and comprises a 2-nucleotide overhang at the 3′-terminus,wherein the sense RNA strand is 23 nucleotides in length and comprises a2-nucleotide overhang at the 3′-terminus, and wherein the dsRNA has a21-nucleotide double-stranded region.
 11. A pharmaceutical compositionfor inhibiting the replication of a (+) strand RNA virus in an mammal,comprising a dsRNA and a pharmaceutically acceptable carrier, whereinthe dsRNA comprises a sense RNA strand consisting of a nucleotidesequence which is substantially identical to at least a part of a3′-untranslated region (3′-UTR) of the (+) strand RNA virus and anantisense RNA strand which is complementary to at least a part of thesense RNA strand, and wherein each strand of the dsRNA is 21 to 24nucleotides in length. 12-13. (canceled)
 14. The pharmaceuticalcomposition of claim 11, wherein the dsRNA comprises a blunt end. 15.The pharmaceutical composition of claim 11, wherein the dsRNA comprisestwo blunt ends.
 16. The pharmaceutical composition of claim 11, whereinthe antisense RNA strand and the sense RNA strand comprise a 3′-terminusand a 5′-terminus, and wherein at least one of said RNA strands comprisea nucleotide overhang of 1 to 3 nucleotides in length.
 17. Thepharmaceutical composition of claim 16, wherein the nucleotide overhangis two nucleotides in length.
 18. The pharmaceutical composition ofclaim 16, wherein the nucleotide overhang is on the 3′-terminus of theantisense RNA strand.
 19. The pharmaceutical composition of claim 16,wherein the dsRNA further comprises a first end and a second end,wherein the first end comprises the 3′-terminus of the antisense RNAstrand and the 5′-terminus of the sense RNA strand, and wherein thesecond end comprises the 5′-terminus of the antisense RNA strand and the3′-terminus of the sense RNA strand, wherein the first end comprises anucleotide overhang on the 3′-terminus of the antisense RNA strand, andwherein the second end is blunt.
 20. The pharmaceutical composition ofclaim 16, wherein the antisense RNA strand is 23 nucleotides in lengthand comprises a 2-nucleotide overhang at the 3′-terminus, wherein thesense RNA strand is 23 nucleotides in length and comprises a2-nucleotide overhang at the 3′-terminus, and wherein the dsRNA has a21-nucleotide double-stranded region.
 21. The pharmaceutical compositionof claim 11, wherein a dosage unit of dsRNA is less than 5 milligram(mg) of dsRNA per kg body weight of the mammal.
 22. The pharmaceuticalcomposition of claim 11, wherein a dosage unit of dsRNA is in a range of0.01 to 2.5 milligrams (mg), 0.1 to 200 micrograms (μg), 0.1 to 100 μgper kilogram body weight of the mammal.
 23. The pharmaceuticalcomposition of claim 11, wherein a dosage unit of dsRNA is less than 25μg per kilogram body weight of the mammal.
 24. The pharmaceuticalcomposition of claim 11, wherein the pharmaceutically acceptable carrieris an aqueous solution.
 25. The pharmaceutical composition of claim 24,wherein the aqueous solution is phosphate buffered saline.
 26. Thepharmaceutical composition of claim 11, wherein the pharmaceuticallyacceptable carrier comprises a micellar structure selected from thegroup consisting of a liposome, capsid, capsoid, polymeric nanocapsule,and polymeric microcapsule.
 27. The pharmaceutical composition of claim11, which is formulated to be administered by inhalation, infusion,injection, or orally.
 28. The pharmaceutical composition of claim 11,which is formulated to be administered by intravenous or intraperitonealinjection.